Template and primer requirements for DNA Pol θ-mediated end joining

Proc Natl Acad Sci U S A. 2018 Jul 24;115(30):7747-7752. doi: 10.1073/pnas.1807329115. Epub 2018 Jul 9.

Abstract

DNA Pol θ-mediated end joining (TMEJ) is a microhomology-based pathway for repairing double-strand breaks in eukaryotes. TMEJ is also a pathway for nonspecific integration of foreign DNAs into host genomes. DNA Pol θ shares structural homology with the high-fidelity replicases, and its polymerase domain (Polθ) has been shown to extend ssDNA without an apparent template. Using oligonucleotides with distinct sequences, we find that with Mg2+ and physiological salt concentrations, human Polθ has no terminal transferase activity and requires a minimum of 2 bp and optimally 4 bp between a template/primer pair for DNA synthesis. Polθ can tolerate a mismatched base pair at the primer end but loses >90% activity when the mismatch is 2 bp upstream from the active site. Polθ is severely inhibited when the template strand has a 3' overhang within 3-4 bp from the active site. In line with its TMEJ function, Polθ has limited strand-displacement activity, and the efficiency and extent of primer extension are similar with or without a downstream duplex.

Keywords: 3′ overhang; alt-NHEJ; base pair; mismatch; template.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Catalytic Domain
  • DNA / biosynthesis
  • DNA / chemistry*
  • DNA / genetics
  • DNA End-Joining Repair*
  • DNA Polymerase theta
  • DNA Primers / chemistry*
  • DNA Primers / genetics
  • DNA Primers / metabolism
  • DNA-Directed DNA Polymerase / chemistry*
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / metabolism
  • HEK293 Cells
  • Humans

Substances

  • DNA Primers
  • DNA
  • DNA-Directed DNA Polymerase